To develop a model system in which we could induce GCRs, we used the rare restriction enzyme I-SceI, whose 18 bp recognition sequence is not normally present in the human or mouse genome, to produce a single DNA DSB within a mammalian cell, based on the hypothesis that improper repair of these breaks could lead to GCRs. This enzyme has been used in a series of elegant studies to produce specific, non-random GCRs mediated by homologous recombination in mammalian cells. We generated a construct that expressed the Herpes simplex virus type I thymidine kinase (TK) gene under the control of the constitutive EF1a promoter, with the recognition sequence for the I-SceI restriction enzyme placed between the EF1a promoter and the TK gene. This pEF1aTK vector was introduced into the U937 cell line, and verified that expression of the TK gene conferred sensitivity to ganciclovir (GCV). We then carried out a series of experiments that utilized the negative selection provided by the expression of TK. Cells were transfected with an I-SceI expression vector and selected with GCV (to select for cells that had lost TK expression). We characterized over 100 independent clones. All of the clones had small deletions and showed evidence consistent with non-homologous end joining (NHEJ), such as microhomology or local sequence inversions. no clones showed evidence of having a GCR. We considered the possibility that the U937 cell line, which has a fairly normal karyotype [47 XX, t(10;11)] may not be an ideal choice for these types of experiments, and that a structurally unstable cell line might be more likely to produce GCR. The NCI 60 cell line panel has been assayed for numerical and structural instability;OVCAR8 showed the highest level of ongoing structural instability. We generated OVCAR-8 subclones that harbored a single copy of the pEF1aTK vector, and transfected these clones with I-SceI expression vectors. However, similar to the results with the U937 clones, all 31 GCV-resistant (GCVR) clones had small interstitial deletions and features of repair via NHEJ. These results were published in 2010. We also considered the possibility that a potential disadvantage of the above approach is that it utilizes a negative selection system, and will generate clones that delete small (i.e. greater than 100bp) portions of the EF1a promoter or TK cDNA, leading to lack of TK expression and GCVR. Therefore, we developed a complementary vector that allowed positive selection. This vector contains a hygromycin phosphotransferase gene (HygroR;confers resistance to hygromycin) preceded by an I-SceI recognition sequence, but no promoter region. We transfected this promoter-less vector into OVCAR 8 cells, and identified clones that integrated a single copy. As anticipated, since the HygroR gene lacks a promoter, these cells are hygromycin sensitive. These cells were then transfected with an I-SceI expression vector, and selected with hygromycin, in the hopes of recovering rare clones that had undergone a GCR, and juxtaposed a promoter from a distant genomic region, thus allowing expression of the HygroR gene, leading to hygromycin resistance. All of the clones analyzed were vector capture events, in which a portion of SV40 regulatory sequences derived from the I-SceI expression vector have become juxtaposed to the hygroR gene. These results were published in 2010. It may not be surprising that we were unable to generate GCRs by inducing a single DNA DSB, as other investigators have concluded that two induced breaks are required to produce a chromosomal translocation, and that the frequency of chromosomal translocations induced by a single DNA DSB in mouse embryonic stem (ES) cells is extraordinarily rare ( less than 5 x 10E08). However, an alternative, error prone NHEJ pathway has recently been described, and has been implicated in chromosomal translocations associated with malignancy. In addition, mice lacking one or more elements of the NHEJ repair pathway (such as Ku70/80, Lig4, or DNA-PKcs) or H2ax are prone to complex chromosomal rearrangements involving cellular proto-oncogenes. Therefore, we began studies to determine if inhibition of normal DNA DSB repair pathway components can lead to GCR after induction of a single DNA DSB. We obtained spontaneously immortalized H2ax-/- and Ku80 -/- murine fibroblast cell lines, and generated clones containing a single copy of the pEF1alphaTK vector. We transfected an I-SceI expression vector into H2ax-/- cells containing a single copy of the pEF1alphaTK vector (clone H2AX8), and selected GCV-resistant clones. The spectrum of DNA rearrangements identified in the H2ax-/- cells was markedly different than in any of the aforementioned studies. Of 26 clones characterized, only 2 were small interstitial deletions. 19 clones had vector capture events, and five clones represented GCRs, two balanced translocations and three chromosomal inversions of 3-78 Mb. We are currently preparing a manuscript that will report these findings. We have also used this same I-SceI based system to determine if simultaneous introduction of and AID-expression vector and I-SceI will produce GCRs. We do this because the AID enzyme induces class switch recombination in the immunoglobulin locus through the induction of DNA double strand breaks. It seems possible that introduction of one break by I-SceI, and a second break by AID, may lead to a GCR, by an error-prone DNA repair process. We are also attempting to move this system from a cell-culture based system to an in vivo system. To do so, we take advantage of the observation that the MLL gene is known to have at least 100 oncogenic partners, and is even weakly oncogenic when fused to a LacZ reporter gene. These findings suggest that MLL fused to many other genes may be oncogenic. We used gene targeting to insert an I-SceI site in the MLL locus. These mice have been crossed to mice that express the ISceI protein in hematopoietic cells, to test the possibility that a GCR involving MLL will be generated in vivo. We reasoned that even if this is a very rare event, it may be amplified in vivo, as cells that undergo an MLL fusion may be oncogenic. Unfortunately, none of over 40 mice have developed leukemia yet. We reasoned that this may be because repair of the I-SceI induced breaks is very efficient. We have now begun to cross these transgenes onto an H2AX deficient background, to determine if a more error-prone DNA repair pathway will result in MLL translocations in vivo.